PROJECT SUMMARY Maintaining mitochondrial integrity is necessary for normal eukaryotic physiology and, not surprisingly, mitochondrial dysfunction is a pathological hallmark of diseases and has been implicated as a primary risk factor for many cancers and neurodegenerative disorders. Critical to mitochondrial function is its dual-membrane architecture which provides appropriate microenvironments that facilitate specific metabolic functions ? such as oxidative phosphorylation ? and allow for otherwise incompatible processes to occur simultaneously inside the cell. Traditionally, research on mitochondria have focused on bioenergetics, but recent studies have begun to shed light on the intricacies and complexities of the mitochondrial proteome and the biogenesis machineries. This is particularly important as >99% of the mitochondrial proteome (~1500 proteins in humans) are encoded by nuclear genes and synthesized by cytosolic ribosomes as precursor proteins (preproteins). These preproteins contain endogenous signals that target them to mitochondria, where they are subsequently translocated across the outer membrane, sorted, compartmentalized, and properly folded by three main protein import machineries: the translocase of the outer mitochondrial membrane (TOM) complex, the mitochondrial translocase of the inner membrane (TIM)-23 complex (TIM23), and the TIM22 complex. These protein import complexes are required for the biogenesis of nearly all mitochondrial proteins and dysregulation poses a significant challenge to maintaining normal mitochondrial physiology. However, a dearth of structural information has precluded a molecular understanding of these processes and the mechanisms by which they perform their critical functions. Under this award, I will develop groundbreaking three-dimensional (3D) electron cryomicroscopy (cryoEM) technologies to pioneer studies of these critically important mitochondrial protein import complexes, providing critical insights into their function and their roles in the disease state. I will utilize targeted biochemical approaches to isolate the TOM, TIM23, and TIM22 complexes from natural sources for high-resolution cryoEM studies. I will then develop novel EM sample preparation, data collection and data processing strategies to yield a suite of high-resolution structures of each of these import machines during active preprotein import. I will then quantify the degree of local and global dynamics within these states through novel atomic modeling strategies as a means to define the conformational landscape. I will then establish in vitro functional assays to test key molecular steps during these processes. Lastly, I will then develop correlated light microscopy and high- resolution electron cryotomography (cryoET) methodologies to determine structure of these import complexes in their native membranes. Through these combined efforts, I will answer fundamental questions pertaining to the overall 3D architecture of these complexes and fully describe the molecular motions necessary for these import machines to import and fold mitochondrial preproteins. Importantly, these methodologies can be extended beyond the immediate scope of this award and be applied ubiquitously across the cryoEM community.